Chemiluminescence detection of NADH and enzyme substrates with immobilized tris(2,2'-bipyridyl)ruthenium (II) and dehydrogenase enzymes
Martin, Alice Fay
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/19869
Description
Title
Chemiluminescence detection of NADH and enzyme substrates with immobilized tris(2,2'-bipyridyl)ruthenium (II) and dehydrogenase enzymes
Author(s)
Martin, Alice Fay
Issue Date
1995
Doctoral Committee Chair(s)
Nieman, Timothy A.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Chemistry, Biochemistry
Language
eng
Abstract
The objective of this work is to couple the sensitivity of tris(2,2$\sp\prime$-bipyridyl)ruthenium(II) (Ru(bpy)$\sb3\sp{2+}$) electrogenerated chemiluminescence (ECL) to the selectivity of dehydrogenase enzymes. Within this objective, methods of reagent immobilization for Ru(bpy)$\sb3\sp{2+}$ and dehydrogenase enzyme have been studied and applied to bioanalytical determinations. Dehydrogenase enzymes employ NAD$\sp+$ as their cofactor and produce NADH in an amount equivalent to reacted analyte. Detection of NADH via the ECL reaction with Ru(bpy)$\sb3\sp{3+}$ is then used to determine analyte concentration.
Several different materials have been investigated as immobilization matrices. Sulfonic cation exchange polymers Nafion (Dupont) and AQ55 (Eastman) have been used for Ru(bpy)$\sb3\sp{2+}$ immobilization to create ECL sensors capable of detecting 5 $\mu$M NADH and lower. The ECL sensor was created by exchanging Ru(bpy)$\sb3\sp{2+}$ into a film of Nafion or AQ55 cast on a platinum electrode. The electrode was then used to generate Ru(bpy)$\sb3\sp{3+}$ which then is available to react with NADH to yield light.
Dehydrogenase immobilization has been carried out in two different formats. First, an enzyme reactor was prepared by covalently crosslinking the dehydrogenase to amino-derivatized glass beads and packing them in a column. The column was then used on-line in a flow injection analysis system with the ECL sensor as a detector. The conditions for optimum ECL signal were: pH (about 6.5), flow rate (2 mL/min), and NAD$\sp{+}$ concentration (1-2.5 mM). Glucose detection was from 5.0 mM to 10 $\sb\mu$M glucose.
The second format entrapped the dehydrogenase in AQ55 to be used in an ECL biosensor. Glucose, L-lactate or alcohol dehydrogenase were mixed with AQ55(D) to form an enzyme layer on top of or near a polymer modified electrode containing Ru(bpy)$\sb3\sp{3+}.$ The enzyme layer was treated with an overlayer of Nafion to protect the polymer film from redissolving in an aqueous solution. Three biosensor designs based on different physical positions of the two layers were evaluated. Using a glucose dehydrogenase based sensor, glucose is detected from 3.0 mM to 10 $\mu$M. Additionally, it has been shown that both NAD$\sp+$ and NADP$\sp+$ may be used as dehydrogenase cofactors where NADPH itself may be detected from 10 nM to 100 $\mu$M.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
